The overall goal of this project is to improve the safety and efficacy of transfusion of stored blood. There is good evidence that red blood cell storage results in loss of functionality and integrity of red blood cells over time and that this contributes to deleterious effects upon blood transfusion. This project, which is a competitive renewal of a previously funded one, is built around the hypothesis that this storage lesion is largely due to dysregulation of nitric oxide homeostasis in the blood. This disruption is due to increased nitric oxide scavenging by cell-free hemoglobin and microparticles that are released during hemolysis in stored blood and diminished nitric oxide production by the newly discovered red cell nitric oxide synthase. In this renewal we will examine the exact mechanisms of loss of NO bioavailability, as well as down-stream effects of this loss, particularly platelet activation. e will also study interactions that occur in a susceptible host receiving the transfusion. A vast array of clinical, biophysical, molecular biology, and biochemical tools will be applied to characterize the nitric oxide storage lesion in vitro in stored blood as well as in chimeric mice models, a canine model, and in human studies. In addition, therapeutics will be explored in these systems that could restore nitric oxide homeostasis by increasing nitric oxide production.